Protein tyrosine phosphorylation is an essential element in signal transduction pathways which control fundamental cellular processes including growth and differentiation, cell cycle progression, and cytoskeletal function. Briefly, the binding of growth factors, or other ligands, to a cognate receptor protein tyrosine kinase (PTK) triggers autophosphorylation of tyrosine residues in the receptor itself and phosphorylation of tyrosine residues in the enzyme's target substrates. Within the cell, tyrosine phosphorylation is a reversible process; the phosphorylation state of a particular tyrosine residue in a target substrate is governed by the coordinated action of both PTKs, catalyzing phosphorylation, and protein tyrosine phosphatases (PTPs), catalyzing dephosphorylation.
The PTPs are a large and diverse family of enzymes found ubiquitously in eukaryotes [Charbonneau and Tonks, Ann. Rev. Cell Biol. 8:463–493 (1993)]. Structural diversity within the PTP family arises primarily from variation in non-catalytic (potentially regulatory) sequences which are linked to one or more highly conserved catalytic domains. In general, soluble cytoplasmic PTP forms are termed non-receptor PTPs and those with at least one non-catalytic region that traverses the cell membrane are termed receptor-like PTPs (RPTPs).
A variety of non-receptor PTPs have been identified which characteristically possess a single catalytic domain flanked by non-catalytic sequences. Such non-catalytic sequences have been shown to include, among others, sequences homologous to cytoskeletal-associated proteins [Yang and Tonks, Proc. Natl. Acad. Sci. (USA) 88:5949–5953 (1991)] or to lipid binding proteins [Gu, et al., Proc. Natl. Acad. Sci. (USA) 89:2980–2984 (1992)], and/or sequences that mediate association of the enzyme with specific intracellular membranes [Frangioni et al., Cell 68:545–560 (1992)], suggesting that subcellular localization may play a significant role in regulation of PTP activity.
Analysis of non-catalytic domain sequences of RPTPs suggests their involvement in signal transduction mechanisms. However, binding of specific ligands to the extracellular segment of RPTPs has been characterized in only a few instances. For example, homophilic binding has been demonstrated between molecules of PTPμ [Brady-Kalnay, et al., J. Cell. Biol. 122:961–972 (1993)] i.e., the ligand for PTPμ expressed on a cell surface is another PTPμ molecule on the surface of an adjacent cell. Little is otherwise known about ligands which specifically bind to, and modulate the activity of, the majority of RPTPs.
Many receptor-like PTPs comprise an intracellular carboxyl segment with two catalytic domains, a single transmembrane domain and an extracellular amino terminal segment [Krueger et al., EMBO J. 9:3241–3252 (1990)]. Subclasses of RPTPs are distinguished from one another on the basis of categories or “types” of extracellular domains [Fischer, et al., Science 253:401–406 (1991)]. Type I RPTPs have a large extracellular domain with multiple glycosylation sites and a conserved cysteine-rich region. CD45 is a typical Type I RPTP. The Type II RPTPs contain at least one amino terminal immunoglobulin (Ig)-like domain adjacent to multiple tandem fibronectin type III (FNIII)-like repeats. Similar repeated-FNIII domains, believed to participate in protein:protein interactions, have been identified in receptors for IL2, IL4, IL6, GM-CSF, prolactin, erythropoietin and growth hormone [Patthy, Cell 61:13–14 (1992)]. The leukocyte common antigen-related PTP known as LAR exemplifies the Type II RPTP structure [Streuli et al., J. Exp. Med. 168:1523–1530 (1988)], and, like other Type II RPTPs, contains an extracellular region reminiscent of the NCAM class of cellular adhesion molecules [Edelman and Crossin, Ann. Rev. Biochem. 60:155–190 (1991)]. The Type III RPTPs, such as HPTPβ [Krueger et al., EMBO J. 9:3241–3252 (1990)], contain only multiple tandem FNIII repeats in the extracellular domain. The Type IV RPTPs, for example RPTPα [Krueger et al. (1990) supra], have relatively short extracellular sequences lacking cysteine residues but containing multiple glycosylation sites. A fifth type of RPTP, exemplified by PTPγ [Barnes, et al., Mol. Cell. Biol. 13:1497–1506 (1993)] and PTPζ [Krueger and Saito, Proc. Natl. Acad. Sci.(USA) 89:7417–7421 (1992)], is characterized by an extracellular domain containing a 280 amino acid segment which is homologous to carbonic anhydrase (CAH) but lacks essential histidine residues required for reversible hydration of carbon dioxide.
FNIII sequences characteristically found in the extracellular domains of Type II and Type III RPTPs comprise approximately ninety amino acid residues with a folding pattern similar to that observed for Ig-like domains [Bork and Doolittle, Proc. Natl. Acad. Sci(USA) 89:8990–8994 (1992)]. Highly conserved FNIII sequences have been identified in more than fifty different eukaryotic and prokaryotic proteins [Bork and Doolittle, Proc. Natl. Acad. Sci. (USA) 89:8990–8994 (1992)], but no generalized function has been established for these domains. Fibronectin itself contains fifteen to seventeen FNIII domain sequences, and it has been demonstrated that the second FNIII domain (FNIII2) contains a binding site for heparin sulphate proteoglycan [Schwarzbauer, Curr. Opin. Cell Biol. 3:786–791 (1991)] and that FNIII13 and FNIII14 are responsible for heparin binding through ionic interactions [Schwarzbauer, Curr. Opin. Cell Biol. 3:786–791 (1991)]. Perhaps the best characterized interaction for a fibronectin FNIII domain involves FNIII10 which is the major site for cell adhesion [Edelman and Crossin, Ann. Rev. Biochem 60:155–190 (1991); Leahy, et al., Science 258:987–991 (1992), Main, et al., Cell 71:671–678 (1992)]. FNIII10 contains the amino acid sequence Arg-Gly-Asp (RGD) which is involved in promoting cellular adhesion through binding to particular members of the integrin superfamily of proteins.
Characteristics shared by both the soluble PTPs and the RPTPs include an absolute specificity for phosphotyrosine residues, a high affinity for substrate proteins, and a specific activity which is one to three orders of magnitude in excess of that of the PTKs in vitro [Fischer, et al., Science 253:401–406 (1991); Tonks, Curr. Opin. Cell. Biol. 2:1114–1124 (1990)]. This latter characteristic suggests that PTP activity may exert an antagonistic influence on the action of PTKs in vivo, the balance between these two thus determining the level of intracellular tyrosine phosphorylation. Supporting a dominant physiological role for PTP activity is the observation that treatment of NRK-1 cells with vanadate, a potent inhibitor of PTP activity, resulted in enhanced levels of phosphotyrosine and generation of a transformed cellular morphology [Klarlund, Cell 41:707–717 (1985)]. This observation implies potential therapeutic value for PTPs and agents which modulate PTP activity as indirect modifiers of PTK activity, and thus, levels of cellular phosphotyrosine.
Recent studies have highlighted aspects of the physiological importance of FITP activity. For example, mutations in the gene encoding a non-receptor hematopoietic cell protein tyrosine phosphatase; HCP, have been shown to result in severe immune dysfunction characteristic of the motheaten phenotype in mice [Schultz, et al., Cell 73:1445–1454 (1993)]. Under normal conditions HCP may act as a suppressor of PTK-induced signaling pathways, for example, the CSF-1 receptor [Schultz, et al., Cell 73:1445–1454 (1993)]. Some PTP enzymes may be the products of tumor suppressor genes and their mutation or deletion may contribute to the elevation in cellular phosphotyrosine associated with certain neoplasias [Brown-Shimer, et al., Cancer Res. 52:478–482 (1992); Wary, et al., Cancer Res. 53:1498–1502 (1993)]. Mutations observed in the gene for RPTPγ in murine L cells would be consistent with this hypothesis [Wary, et al., Cancer Res. 53.1498–1502 (1993)]. The observation that the receptor-like PTP CD45 is required for normal T cell receptor-induced signalling [Pinget and Thomas, Cell 58:1055–1065 (1989)] provides evidence implicating PTP activity as a positive mediator of cellular signalling responses.
Normal cells in culture exhibit contact inhibition of growth, i.e., as adjacent cells in a confluent monolayer touch each other, their growth is inhibited [Stoker and Rubin, Nature 215:171–172 (1967)]. Since PTKs promote cell growth, PTP action may underlie mechanisms of growth inhibition. In Swiss mouse 3T3 cells, a phosphatase activity associated with membrane fractions is enhanced eight-fold in confluent cells harvested at high density as compared to cells harvested from low or medium density cultures [Pallen and Tong, Proc. Natl. Acad. Sci. (USA) 88:6996–7000(1991)]. This elevated activity was not observed in subconfluent cell cultures brought to quiescence by serum deprivation. The enhanced phosphatase activity was attributed to a 37 kD protein, as determined by gel filtration, but was not otherwise characterized. Similarly, PTPs have been directly linked to density arrest of cell growth; treatment of NRK-1 cells with vanadate was able to overcome density dependent growth inhibition and stimulate anchorage independent proliferation, a characteristic unique to transformed, or immortalized, cells [Klarland, Cell 41:707–717 (4985); Rijksen, et al., J. Cell Physiol. 154:343–401 (1993)].
In contrast to these observations, PCT Publication No. WO 94/03610 discloses a transmembrane PTP, termed PTP35, the steady state mRNA level of which was observed to be at a maximum in actively growing cells. Little or no PTP35 mRNA expression was detected in confluent cell. This mode of regulation was also observed in mouse 3T3 cells. Thus, two RPTPs in the same cell type apparently participate in opposing processes, with one (PTP35) contributing to cellular growth and the other (the 35 kD PTP of Pallen and Tongs) contributing to cellular quiescence.
Interestingly, transcription of Type II RPTP LAR messenger RNA has been demonstrated to be upregulated in confluent fibroblast cell culture [Longo, et al., J. Biol. Chem. 268:26503–26511 (1993)]. LAR is proteolytically processed to generate a mature protein that is a complex of two non-covalently associated subunits, one containing the majority of the cell adhesion molecule-like extracellular domain [Yu, et al., Oncogene 7:1051–1057 (1992); Streuli, et al., EMBO J. 11:897–907 (1992)] and which is shed as cells approach confluence [Streuli, et al., EMBO J. 11:897–907 (1992)]. These observations lead to speculation regarding PTP involvement in modulation of cytoskeletal integrity, as well as other related cellular phenomena such as transformation, tumor invasion, metastasis, cell adhesion, and leukocyte movement along and passage-through the endothelial cell layer in inflammation. The therapeutic implications are enormous for modulators of PTP activity which are capable of regulating any or all of these cellular events.
There thus exists a need in the art to identify members of the PTP family of enzymes and to characterize these proteins in terms of their amino acid and encoding DNA sequences. Such information would provide for the large scale production of the proteins, allow for identification of cells which express the phosphatases naturally and permit production of antibodies specifically reactive with the phosphatases. Moreover, elucidation of the substrates, regulatory mechanisms, and subcellular localization of these PPs would contribute to an understanding of normal cell growth and provide information essential for the development of therapeutic agents useful for intervention in abnormal and/or malignant cell growth.